I am measuring insulin from human plasma samples using pre-coated ELISA plates. It's written in the instruction to first add samples (25ul) then the detection antibody (100ul). Just wondering is it alright to add detection antibody before adding sample? As adding the small volume (25ul plasma) to the great volume (100ul detection antibody) seems a better practice.
Thanks!
I feel the assay format for insulin detection is sandwich in nature. Here detection antibody may be the enzyme labelled antibody. If it is so than the assay procedure provided by the manufacturer is alright. If detection antibody is a labelled one and if you increase its volume by your own than there is chances to increase NSB in assay.
With best wishes
Hi Tulsidas,
Thank you for your reply. Yes the assay is sandwich in nature, and both volumes for detection antibody and samples are specified in the instruction manual. I am just wondering is it alright for me to perform the 2 steps in a different order, as adding the plasma into the detection body can make sure no sample got stuck in the pipette tips (by pipetting up and down).
Regards
According to the instructions, are both the sample and the detection antibody incubated simultaneously?
In this case I think that the order of addition of them will not make a big difference, although in general I prefer to follow manufacturer instructions. The advantage of adding the conjugate before is not clear for me...I understand your idea about better mixing, but I guess the impact of mixing the reagents in a different order is not so important.
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